Akiko Hayashi

PKCnu, a new member of the protein kinase C family, composes a fourth subfamily with PKCmu.

Members of the protein kinase C (PKC) family of serine/threonine kinases are thought to play critical roles in the regulation of cellular differentiation and proliferation in many cell types. An additional member of the PKC family was identified through human expressed sequence tag (EST) database search and its full length cDNA was isolated. Sequence analysis revealed that the predicted translation product was composed of 890 amino acid residues and that the protein has 77.3% similarity to human PKC mu (PKCmu) and 77. 4% similarity to mouse PKD (the mouse homolog of PKCmu). We designated the new member as protein kinase C nu (PKCnu). The PKCnu messenger RNA was ubiquitously expressed in various tissues when analyzed by Northern blots and reverse transcriptase-coupled polymerase chain reaction (PCR) analyses. The chromosomal location of the gene was determined between markers WI-9798 and D2S177 on chromosome 2p21 region by PCR-based methods with both a human/rodent monochromosomal hybrid cell panel and a radiation hybrid mapping panel.

Highly conserved linkage homology between birds and turtles: Bird and turtle chromosomes are precise counterparts of each other

The karyotypes of birds, turtles and snakes are characterized by two distinct chromosomal components, macrochromosomes and microchromosomes. This close karyological relationship between birds and reptiles has long been a topic of speculation among cytogeneticists and evolutionary biologists; however, there is scarcely any evidence for orthology at the molecular level. To define the conserved chromosome synteny among humans, chickens and reptiles and the process of genome evolution in the amniotes, we constructed comparative cytogenetic maps of the Chinese soft-shelled turtle (Pelodiscus sinensis) and the Japanese four-striped rat snake (Elaphe quadrivirgata) using cDNA clones of reptile functional genes. Homology between the turtle and chicken chromosomes is highly conserved, with the six largest chromosomes being almost equivalent to each other. On the other hand, homology to chicken chromosomes is lower in the snake than in the turtle. Turtle chromosome 6q and snake chromosome 2p represent conserved synteny with the chicken Z chromosome. These results suggest that the avian and turtle genomes have been well conserved during the evolution of the Arcosauria. The avian and snake sex Z chromosomes were derived from different autosomes in a common ancestor, indicating that the causative genes of sex determination may be different between birds and snakes.

Cloning, expression analysis, and chromosomal localization of HIP1R, an isolog of huntingtin interacting protein (HIP1)

AbstractHuntington disease (HD) is an inherited neurodegenerative disorder which is associated with CAG expansion in the coding region of the gene for huntingtin protein. Recently, a huntingtin interacting protein, HIP1, was isolated by the yeast two-hybrid system. Here we report the isolation of a cDNA clone for HIP1R (huntingtin interacting protein-1 related), which encodes a predicted protein product sharing a striking homology with HIP1. RT-PCR analysis showed that the messenger RNA was ubiquitously expressed in various human tissues. Based on PCR-assisted analysis of a radiation hybrid panel and fluorescence in situ hybridization, HIP1R was localized to the q24 region of chromosome 12.

Highly reproductive Escherichia coli cells with no specific assignment to the UAG codon.

Escherichia coli is a widely used host organism for recombinant technology, and the bacterial incorporation of non-natural amino acids promises the efficient synthesis of proteins with novel structures and properties. In the present study, we developed E. coli strains in which the UAG codon was reserved for non-natural amino acids, without compromising the reproductive strength of the host cells. Ninety-five of the 273 UAG stop codons were replaced synonymously in the genome of E. coli BL21(DE3), by exploiting the oligonucleotide-mediated base-mismatch-repair mechanism. This genomic modification allowed the safe elimination of the UAG-recognizing cellular component (RF-1), thus leaving the remaining 178 UAG codons with no specific molecule recognizing them. The resulting strain B-95.ΔA grew as vigorously as BL21(DE3) in rich medium at 25–42°C, and its derivative B-95.ΔAΔfabR was better adapted to low temperatures and minimal media than B-95.ΔA. UAG was reassigned to synthetic amino acids by expressing the specific pairs of UAG-reading tRNA and aminoacyl-tRNA synthetase. Due to the preserved growth vigor, the B-95.ΔA strains showed superior productivities for hirudin molecules sulfonated on a particular tyrosine residue, and the Fab fragments of Herceptin containing multiple azido groups.

Reassignment of a rare sense codon to a non-canonical amino acid in Escherichia coli

The immutability of the genetic code has been challenged with the successful reassignment of the UAG stop codon to non-natural amino acids in Escherichia coli. In the present study, we demonstrated the in vivo reassignment of the AGG sense codon from arginine to l-homoarginine. As the first step, we engineered a novel variant of the archaeal pyrrolysyl-tRNA synthetase (PylRS) able to recognize l-homoarginine and l-N6-(1-iminoethyl)lysine (l-NIL). When this PylRS variant or HarRS was expressed in E. coli, together with the AGG-reading tRNAPylCCU molecule, these arginine analogs were efficiently incorporated into proteins in response to AGG. Next, some or all of the AGG codons in the essential genes were eliminated by their synonymous replacements with other arginine codons, whereas the majority of the AGG codons remained in the genome. The bacterial hosts ability to translate AGG into arginine was then restricted in a temperature-dependent manner. The temperature sensitivity caused by this restriction was rescued by the translation of AGG to l-homoarginine or l-NIL. The assignment of AGG to l-homoarginine in the cells was confirmed by mass spectrometric analyses. The results showed the feasibility of breaking the degeneracy of sense codons to enhance the amino-acid diversity in the genetic code.

Structure, expression profile and chromosomal location of an isolog of DNA-PKcs interacting protein (KIP) gene.

A novel DNA-PKcs interacting protein, KIP (kinase interacting protein), was recently isolated using a two-hybrid analysis which showed a significant homology to calcineurin B. We found other ESTs showing significant similarity to KIP gene in the dbEST database and isolated a cDNA clone which encodes a 187 amino acid polypeptide from a human fetal brain cDNA library. This protein (termed KIP2 for kinase interacting protein 2) has sequence homology to KIP (46% identical and 64% similarity). RT-PCR analysis showed that the messenger RNA was ubiquitously expressed in various human tissues. Based on PCR-based analysis with a radiation hybrid cell panel and fluorescence in situ hybridization, the gene was localized to the q24 region of chromosome 15.

Cloning and expression profile of mouse and human genes, Rnf11/RNF11, encoding a novel RING-H2 finger protein.

The RING finger (C3HC4-type zinc finger) is a variant zinc finger motif presents in a new family of proteins. A new member of the RING finger family was identified and its cDNA structures were determined in human and mouse. The predicted protein consisting of a 144 amino acid residues is very conservative between the two species and contains a canonical RING-H2 finger motif (C3H2C2) at the carboxyl-terminal region. The genes were designated as RNF11/Rnf11 for RING finger protein 11. A single 2.4-kb transcript of mouse Rnf11 was ubiquitously expressed in various fetal and adult mouse tissues by the Northern blot analysis. The human RNF11 gene was mapped on chromosome 1p31-p32 region, where frequent alterations have been observed in T-cell acute lymphoblastic leukemia.

Cloning, tissue expression, and chromosomal assignment of human MRJ gene for a member of the DNAJ protein family.

AbstractThe DnaJ protein family consists of proteins with a highly conserved amino acid stretch called the “J - domain”. A cDNA clone encoding a new protein with a J-domain was isolated from a human fetal brain cDNA library. This new member of the DnaJ family of 241 amino acid residues showed 94% identity with mouse Mrj (accession number, AF035962) and 71% identity with mouse Msj-1 (accession number, U95607) along its entire sequence. Reverse transcription - coupled polymerase chain reaction (RT-PCR) analysis showed the messenger RNA was ubiquitously expressed in various human tissues. The chromosomal location of the gene was determined by PCR-based analyses with both a human/rodent monochromosomal hybrid cell panel and a radiation hybrid panel to map on chromosome 11q25 region.

Structure, expression profile, and chromosomal location of a mouse gene homologous to human DNA-PKcs interacting protein (KIP) gene

Genome Research Group, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan Division of Biology and Oncology, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan Biotechnology and Medical Engineering Field, Aisin Cosmos R&D Co., Ltd., 5-2-11 Sotokanda, Chiyoda-ku, Tokyo 101-0021, Japan Laboratory of Animal Genetics, School of Bioagricultural Sciences, Nagoya University, Furocho, Chikusa-ku, Nagoya 464-8601, Japan

Mouse cdc21 only 0.5 kb upstream from dna-pkcs in a head-to-head organization: an implication of co-evolution of ATM family members and cell cycle regulating genes.

The catalytic subunit of the DNA-dependent protein kinase (DNAPKcs) is a member of the ATM family, which in turn is a branch of the phosphatidylinositol 3-kinase (PI3K) superfamily (Hartley et al. 1995). The ATM family consists of relatively large proteins, all of which have motifs found in PI3Ks’ catalytic domains at their carboxy-terminal region. Many of the family members have been found to be involved in DNA repair, cell-cycle checkpoint control, and cell-cycle transition control (Zakian 1995). Furthermore, the ATM gene itself was demonstrated to be the responsible gene for the genetic disorder ataxia telangiectasia (AT) with a wide spectrum of clinical manifestations including hypersensitivity to ionizing radiation and radiomimetic drugs (Savitsky et al. 1995). The hypersensitivity is due to the dysfunction of radiation-induced checkpoint control in AT cells (Shiloh 1995). DNA-PKcs with Ku components catalyzes double-stranded broken DNA-dependent phosphorylation of proteins (Gottlieb and Jackson 1993), suggesting that DNA-PK is a surveyor of damaged DNA. Although several biochemical and mapping studies suggested that the mouse geneDna-pkcsmight be thescid-responsible gene, the exact nature of the mutation remained unknown. Very recent reports described a candidate mutation of the gene (Blunt et al. 1996; Danska et al. 1996), and we made the definitive identification of the scid mutation inDna-pkcs gene (Araki et al. 1997). The last study proved that the truncation of the DNA-PK csin the carboxy-terminal kinase domain is indeed responsible for the scid mutation. In the following course of studying the mouse promoter region of Dna-pkcs, we obtained several genomic clones that corresponded to the 5 8 end portion of the gene. Nucleotide sequencing of these clones confirmed a homology stretch to the previously determined 5 8 end sequence of the cDNA of Dna-pkcs. The first exon with the putative initial methionine of Dna-pk cs (Araki et al. 1997) was identified, and the successive upstream sequence was also determined (Fig. 1; DDBJ/EMBL/GenBank accession number AB000629). The TFD transcription factor DNA binding site database search with this sequence suggested a variety of ciselemental motifs for transcription factors. In Fig. 1, only basic motifs such as two Sp1 boxes and three CCAAT boxes are indicated. The nucleotide sequence databases search unexpectedly revealed a complete homology stretch to Cdc21(Kimura et al. 1995) in the promoter region. This homologous sequence is also indi-